The isolation of histidine tRNA from Thermus thermophilus and the study of its primary structure and interaction sites with homologous aminoacyl-tRNA synthetase

The structure of histidine tRNA (tRNA) is different from tRNA structures of other amino acids specificities by the presence of an extra nucleotide on the 5’-end (G-1). To study the molecular mechanism of interaction of tRNA with histidyl-tRNA synthetase the purification method of T.thermophilus tRNA was elaborated and the primary structure of isoacceptor form of tRNA 1 His was studied. Individual tRNA was isolated from the crude tRNA, using the combination of low pressure chromatography on benzoyl-DEAE-cellulose (BD-cellulose) and DEAE Toyopearl 650 with high pressure liquid chromatography on DEAE 5PW and Ultrapore C8 columns. The primary structure of T.thermophilus tRNA 1 His was determined using the method of rapid gel-sequencing. The studied structure is different from Escherichia coli tRNA in 23 positions. The interaction sites of T.thermophilus tRNA with histidyl-tRNA synthetase were investigated using the method of chemical modification by ethylnitrosourea. It was shown that histidyl-tRNA synthetase protects the following phosphates of tRNA from alkylation by ethylnitrosourea: 8 – between acceptor and D-stem; 27, 28, 29 – from 5’-side of anticodon stem; phosphate 34 in anticodon and phosphates 67, 68 from 3’-side of acceptor stem. All the revealed sites of tRNA are localized on one side of three-dimensional structure of tRNA in the same one where the variable stem side is. D-stem is located on the opposite side and does not interact with the enzyme.

Introduction.The fidelity of protein biosynthesis depends on correct aminoacylation of tRNA by aminoacids.This reaction is catalyzed by aminoacyl-tRNA synthetases which recognize the aminoacid and tRNA specifically.At present there is considerable progress in solving the problem of how aminoacyl-tRNA synthetase can specifically recognize cognate tRNAs from a pool of various tRNA species sharing a similar tetriary structure [1,2].Histidine tRNA is unique in possessing an extra nucleotide G-1 at the 5'-end, which forms the additional base-pair with the discriminatory residue 73, thus the acceptor stem of tRNA His is one nucleotide base-pair longer than other tRNAs.tRNA His of prokaryotes and organelles have C73 as their discriminator base, while eukaryotes have A73 and G73 [3].
The study of aminoacylation of Escherichia coli tRNA His transcripts in vitro proved that the G1 -C73 base-pair is the most important determinant of the recognition by histidyl-tRNA synthetase, while anticodon plays a less expressed role [4].Contrariwise, the investigation of aminoacylation of tRNA transcripts from yeast proved that the contribution of anticodon into tRNA recognition by histidyl-tRNA synthetase is bigger in comparison with the role of discriminatory basis [5].At present the crystals of E.coli and T.thermophilus tRNA synthetase complexes are obtained with low molecular substrates -histidine and histidyl -adenylate, using the X-ray structural analysis their structure is determined, and the mechanism of histidine activation is suggested [6][7][8][9].On the basis of charges distribution investigation on the surface of T.thermophilus histidyl-tRNA synthetase and the comparison of these results with the data for other aminoacyl-tRNA synthetases of IIa class, the model of tRNA recognition by enzyme is suggested [8].Also the crystals of tRNA His complex with T.thermophilus histidyl-tRNA synthetase were obtained [7], but the structure of this complex was not defined yet.Thus, the study of tRNA His interaction with histidyl-tRNA synthetase in the solution is of significant interest.
This work describes the isolation of T.thermophilus tRNA His , the definition of its primary structure and the study of interaction sites with histidyl-tRNA synthetase in the solution.
The cells of extreme thermophile T.thermophilus HB-27 were grown in the fermenter with the volume of 300 liters in the medium, containing peptone (2%), yeast extract (1%) and NaCl (0.2%) at the temperature of 74-76°C and intensive aeration with the speed of 200 rpm.The cells were collected by centrifugation.
Crude tRNA was obtained by phenol extraction of RNA from the biomass with the subsequent deproteinization by mixtures of phenol:chlorophorm (1:1) and chlorophorm:isoamyl alcohol (9:1) [10].To purify the RNA from proteins and contaminations of nucleotide and polysaccharide nature, chromatography was used on DEAE-cellulose, equilibrated by 0.0125 M tris-HCl, pH 7.5.At first, the contaminations were eluated by 0.15 M NaCl, and then by 0.25 M NaCl in the same buffer, tRNA from the column was eluated by 1 M NaCl, precipitated by 2.5 of the volume of cooled ethanol in the presence of 2% of potassium acetate.The sediment of tRNA was collected by centrifugation, then washed by 70% etanol and dried.
The individual tRNA His was isolated from crude tRNA, using chromatography on benzoyl-DEAE-cellulose (BD-cellulose), DEAE Toyopearl 650 and high pressure liquid chromatography (HPLC) on the columns of DEAE 5PW and Ultrapore C8 ("Gold System" equipment of "Beckman" company, USA).
T. thermophilus histidyl-tRNAsynthetase was obtained in the homogeneous state using the method described before [7].
The acceptor activity of tRNA His on different stages of purification was defined according to the maximum level of forming 14 C-histidyl-tRNA.Standard reaction mixture in the volume of 0.25 ml contained 0.1 tris-HCl, pH 7.6, 10 mM MgCl 2 , 5 mM ATP, 0.02 mM 14 C-histidine, 0.4 mg of crude preparation of T. thermophilus aminoacyl-tRNA synthetases and 0.05 ml of the tRNA solution from the investigated fractions.The reaction mixture was incubated for 7 minutes at 65°C, the reaction was stopped by adding 0.5 ml of cooled 10% TCA.The sediments of aminoacyl-tRNA were applied to GFC filters, washed by 2% TCA, dried and the radioactivity was measured by liquid scintillation spectroscopy.
The primary structure of tRNA His was studied, using two methods of sequencing the labeled tRNA: specific chemical degradation [17] and enzymatic sequencing, based on the hydrolysis of tRNA by specific endonucleases [16,17].
Enzymatic hydrolysis of T. thermophilus tRNAHis was performed for 15 minutes at 55°C in 5 ml of 20 mM Na-citrate, pH 5.0, 1 mM EDTA, 7 M urea were used for ribonucleases of T1 and PhyM; 20 mM Na-citrate, pH 3.5, 1 mM EDTA, 7 M urea were used for U2; 20 mM Na-citrate, pH 5.0, 1 mM EDTA were used for ribonuclease from B.cereus; 10 mM Na-phosphate, pH 6.5 and 4.2 M urea were used for ribonuclease CL3.tRNA fragments were separated by electrophoresis in 12.5% and 20% PAAG in the presence of 8 M urea.
The alkylation of labeled tRNA His and its complex with histidyl-tRNA synthetase was performed using ethylnitrosourea under conditions, stabilizing three-dimensional structure of tRNA and at the same time favouring the formation of specific complex tRNA His -histidyl-tRNA synthetase [18].The reaction mixture in the final volume of 25 ml contained 50 mM tris-HCl, pH 7.9, 5 mM MgCl 2 , 2.5 mM 2-mercaptoethanol, 0.8 mM tRNA His , 3.2 mM histidyl-tRNA synthetase.Ethylnitrosourea was added to the reaction mixture as a saturated ethanolic solution (2.5 ml).In control experiments, pure ethanol was used in place of the ethylnitrosourea solution.
The modification reaction was carried out at 37°C for 2 hours and stopped by adding 3 ml 3 M sodium acetate, pH 5.5.The aminoacyl-tRNA synthetases were removed by phenol extraction and tRNA was precipitated by adding three volumes of ethanol.For more complete precipitation, 10 mg of glycogen were added to the mixture.
The alkylation of tRNA under denaturating conditions was performed in 25 ml 0.3 M Na-cacodylate buffer, pH 8.0, containing 2 mM EDTA at 80°C for 2 min.
The cleavage of the polynucleotide chain at the modified residues was carried out by incubation in 10 ml 0,1 M tris-HCl pH 9,0 at 55°C for 5 min.The samples of hydrolyzed tRNA were analyzed by gel-electrophoresis in 12.5% PAAG in 5 mM tris-borate buffer, pH 8.3, containing 1 mM EDTA and 8 M urine.The electrophoretic bands were assigned by comparison with the T1 -ribonuclease partial digest of tRNAHis.Autoradiograms were scanned on the "UltraScan XL" densitometer ("LKB", Sweden).
Results and Discussion.The isolation of individual tRNA His from T. thermophilus.tRNA His was isolated from crude tRNA, using the combination of low pressure chromatography on BD-cellulose and DEAE Toyopearl 650 with HPLC on columns DEAE 5PW and Ultrapore C8.
Chromatography on BD-cellulose.4 g of crude tRNA from T. thermophilus were applied to the BD-cellulose column (5 x 60 cm), equilibrated by 50 mM sodium-acetate buffer, pH 4.5, containing 0.35 M NaCl and 10 mM MgCl 2 .Elution was carried out with concave gradient of the NaCl from 0.45 M (buffer A, 4.0 liter) to 1.5 M (buffer B, 2.0 liter).Concave form of the salt gradient was produced using gradient vials of different diameter (D A /D B = 2/1).The application of such a gradient form results in the decrease of the total gradient volume in 40% and in the corresponding speeding of the process.Moreover, the advantages of this gradient in the comparison with the linear one are in the fact that in the beginning of chromatography, when the main mass of material is eluted from the column, small steep slope of gradient is created, and in the end of chromatography, when "tail" drags on, the gradient becomes steeper and the material is eluted more compactly.NaCl concentration in the mixer after the output of volume V(C V ) was calculated according to the formula [ 19].
where C A and C B -initial concentrations of eluent in the reservoir and mixer; V A , V B -initial volumes of the liquid in vessels; S A , S B -squares of vessels cross-section.
Elution speed was 120 ml/h and fractions with volume of 16 ml were collected.After reaching 1.5 M salt concentration the elution was continued with .5 M NaCl in 10% ethanol.Acceptor activity of tRNA for histidine was assayed as described above.Fig. 1 presents typical chromatography on BD-cellulose, which shows that tRNA His was found in the fractions 270-340.These fractions were pooled and precipitated with 2.5 volumes of ethyl alcohol, dried and kept at -20°C.
Chromatography on DEAE Toyapearl 650.350 mg of tRNA, obtained after the chromatography on BD-cellulose, were applied to the column of DEAE Toyapearl 650 (2.6 x 25 cm), equilibrated with 10 mM sodium-acetate buffer, pH 4.5, containing 0.2 M NaCl, 10 mM MgCl 2 and 10% isopropanol.Elution was carried out with linear gradient of NaCl from 0.2 to 0.3 M (2 x 800 ml).Elution speed was 150 ml/h, and fractions volume -15 ml.The fractions containing tRNA His were pooled, precipitated by ethanol and dried.The obtained preparation contained about 30% of tRNA His .
Further purification of tRNA His was carried out using HPLC Gold System ("Beckman", USA).
Chromatography on the column of DEAE 5PW.The following buffer solutions were used to perform chromatography: buffer A -50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 10 mM MgCl 2 and 10% isopropanol; buffer B -the same, but with 1 M NaCl.The increase of NaCl concentration in the chromatography process was in agreement with the curve number 4, programmed in Gold System.The chromatographic fractions were manually collected, and acceptor activity tRNA His was assayed.Retention time of tRNA His on the column was 41 minutes.
Chromatography on the Ultrapore C8 column.The last step of tRNA His purification was the chromatography on the high-pressure column Ultrapore C8.Buffer A contained 50 mM ammonium acetate, and 10 mM MgCl 2 , buffer B -10% isopropanol.The gradient was performed in the same way as in case of chromatography on DEAE 5PW column.The results of tRNA His chromatography on Ultrapore C8 column are shown in Fig. 2. On this purification step two isoacceptor tRNA His were isolated with yield 2.5 and 1.7 mg for RNA 1 His and RNA 2 His , respectively.Purity of obtained preparations, checked by electrophoresis in 8% PAAG with 8 M urea, was not less than 98%.Thus, the method of isolating isoacceptor tRNA His developed by us, allows obtaining highly purified tRNAs, suitable both for biochemical researches and structural work.
The study of the primary tRNA 1 His structure.Before studying the interaction sites of T.thermophilus tRNA 1 His with cognate aminoacyl-tRNA synthetase, it was necessary to define the primary structure of this tRNA.For this aim two complementary methods were used: the method of rapid gel-sequencing according to Pitty [15] and the method of specific nuclease  hydrolysis [17].As a result of conducted researches the complete nucleotide sequence of T.thermophilus tRNA 1 His was defined (Fig. 3).The comparison of primary structure of the studied tRNA 1 His with the structure of E.coli tRNA His reveals rather a great similarity between these macromolecules.Differences were found in 23 positions: eight -in the acceptor stem, two -in D-loop, one -between D-stem and anticodon stem, three -in the anticodon stem, threein the anticodon loop, two -in the T-loop (Fig. 3).The obtained data of the primary structure of T.thermophilus tRNA 1 His show that it has extra G-1 nucleotide on the 5'-end and extra base pair G-1 -C73 in the acceptor stem.These features of tRNA structure are typical for all the known tRNA His from prokaryotes [3].

The study of tRNA 1
His interaction with histidyl-tRNA synthetase by the method of chemical modification.The interaction sites of T.thermophilus tRNA 1 His with the cognates synthetase were investigated using the method of chemical modification by ethylnitrosourea.It was established previously that the alkylation of tRNA phosphate groups in the contact sites with the enzyme is complicated [18].
Typical experiment with 3'-labeled T. thermophilus RNA 1 His is shown in Figure 4.In the presence of histidyl-tRNA synthetase, several bands are nearly suppressed or are strongly reduced, suggesting that the corresponding phosphate groups are protected by the enzyme from alkylation.
The extent of phosphate alkylation was measured by densitometry of the autoradiograph and relative reactivities of phosphates of tRNA 1 His , which is in the complex with the enzyme, were calculated in comparison with the free tRNA.The data of these experiments are shown in Figure 5 and summarized on cloverleaf representation of tRNA in Figure 6.Presence of histidyl-tRNA synthetase resulted in strong protection (40 -70 % decrease in band density ) of phosphates in the acceptor stem (67 and 68) and in the anticodon stem (27,28,29).Weaker protection (20 -40 % decrease in band density) was observed in the corner of the acceptor and D-stems ( phosphate 8 ) and in the anticodon loop (phosphate 34).
It is worth mentioning that all the defined tRNA His sites are on one side of three dimensional tRNA structure, namely, on the side of the variable loop, while D-stem is located on the opposite side of the molecule and it does not interact with the enzyme.Thus, obtained data suggest that the interaction of histidyl-tRNA synthetase with the homologous tRNA shows a recognition pattern characteristic of class 11 aminoacyl-tRNA synthetases [20].
A footprinting technique using phosphorotioate-containing RNA transcripts has been applied to identify contacts between E.coli tRNA His and its cognate aminoacyl-tRNA synthetase [21].However, in the mentioned work the protection of some sites which evidently do not coincide with the expected complex structure on the basis of X-ray structural Fig. 4. Separation in 12. % PAAG of the hydrolysis products of 3'-labeled tRNA His from T.thermophilus, alkylated by ethylnitrosourea in the conditions, denaturating (I) and stabilizing (3, 5) three dimensional tRNA structure, as well as in the presence of histidyl-tRNA synthetase (7); brackets mark the phosphates, the level of modification of which decreases significantly in the presence of histidyl-tRNA synthetase.Corresponding control incubations (4,6,8) were carried out in the absence of ethylnitrosourea; 2 -tRNA His , partially hydrolyzed by T1 nuclease.The stripes enumeration corresponds to the phosphates in the tRNA His structure.analysis (in particular, sites of D-loop), was revealed besides tRNA sites, which unambiguously interact with the enzyme (e.g.5'-end of anticodon stem).Obtained artifacts are explained by known disadvantages of the method, used by the authors [22].The results, presented by us concerning the protection of phosphate acid residues of tRNA His from the chemical modification in the presence of homologous synthetase are in good agreement with the previously suggested model of tRNA His complex with histidyl-tRNA synthetase from T.thermophilus [8].According to this model tRNA His interacts with both subunits of the enzyme.Here three main contact areas of tRNA with the protein can be distinguished: tRNA acceptor stem, interacting with the catalytic domain of enzyme; core of tRNA, which has a contact with the catalytic part of another enzyme subunit and finally, anticodon stem, interacting with the C-end of protein domain.We did not obtain the data on reactivity phosphates of tRNA CCA-end due to the known methodical limitations (non-quantitative precipitation of short tRNA fragments by ethanol).At the same time the protection of phosphates 67 and 68 is clearly evident in the acceptor stem (Figure 6e, 6).In the model of tRNA His complex with T. thermophilus histidyl-tRNA synthetase the residue of phosphoric acid 67 interacts with the conservative Arg7 and Arg74 in protein.The importance of this interaction is proved by the mutagenesis date, obtained on the histidyl-tRNA synthetase from E. coli, where the mutation of Arg7 resulted in 50-fold decrease of K M for tRNA in the aminoacilation reaction [23].The phosphate 8, partially protected by histidyl-tRNA synthetase from alkylation, according to the model, should interact with the amino acid residues of the loop 96-98.The largest interaction zone of tRNA His with the enzyme is the anticodon arm where, according to our biochemical data, the phosphates 28-30 in the anticodon stem and a phosphate 34 in the anticodon interact with the protein.
For the interaction with the phosphates of anticodon stem in the complex model, there is a cluster of positively charged amino acids [8], formed by the amino acid residues of the loop b13-b14 and the residues, which are included in the composition of alpha-helix a14.To obtain a more complete picture of contacts of C-end domain with anticodon stem tRNA His , as well as to get clear details of the interaction of these macromolecules on the atomic level it is necessary to obtain crystallographic data of the structure of their complex.

Fig. 1 .
Fig. 1.The chromatography of tRNA from T. thermophilus on the column with BD-cellulose: 1-optic density at 260 nm; 2 -activity of tRNA His ; 3gradient of NaCl concentration.

Fig. 2 .
Fig. 2. The purification of tRNA His from T.thermophilus on the Ultrapore column C8.The vertical lines show the fractions which were collected in the course of chromatography.The output time of tRNA His from the column is 20.3 minutes (fraction 2, purification degree 95%) and 23.7 minutes (fraction 4, purification degree 80%).

Fig. 3 .
Fig. 3. Cloverleaf representation of tRNA His from T thermophilus.The triangles mark nucleotides which differ from tRNA His from E. coli.

Fig. 5 .
Fig. 5. Relative reactivities of the tRNA 1 His from T.thermophilus phosphates at the alkylation by ethylnitrosourea in the presence of histidyl-tRNA synthetase.R i are the ratios between the intensities of the corresponding electrophoretic bands of the tRNAHis alkylation patterns in the presence of the enzyme and free tRNA His ; Np -phosphates numbers.

Fig. 6 .
Fig. 6.Nucleotide sequence of tRNA His from T. thermophilus in the form of a cloverleaf: arrows show phosphates of tRNA, which are completely (full arrow) and partially (interrupted arrow) protected by the homologous aminoacyl-tRNA synthetase from the alkylation by ethylnitrosourea.